Comparison of Isoenzyme Pattern of Echinococcus granulosus sensu stricto (G1-G3) and E. canadensis (G6/G7) Protoscoleces

Background: Different genotypes of Echinococcus granulosus s.l. infect humans and ungulate animals, causing cystic echinococcosis. Simultaneous isoenzyme, as well as molecular characterizations of this parasite, has not yet been investigated in Iran. The present study aimed to evaluate the isoenzyme pattern of the E. granulosus s.s. and E. canadensis genotypes in Iran. Methods: A total of 32 (8 humans and 24 animals) cystic echinococcosis cysts were isolated from Shiraz, Tehran, Ilam, and Birjand from May 2018 to December 2020. The DNAs were extracted and their genotypes were determined by molecular methods. Enzymes were extracted from the cysts and subjected to polyacrylamide gel electrophoresis. The activities of G6PD, MDH, ME, NH1, and ICD were examined in the cyst samples using isoenzyme method and compared it with the genotyping findings. Results: DNA sequence analysis of the samples showed that the specimens contained 75% E. granulosus s.s. (G1) and 25% E. canadensis (G6) genotypes. The isoenzyme pattern of ICD in both genotypes produced a six-band pattern with different relative factors. The G6PD also produced two bands with different relative migrations in both genotypes. The MDH and NH1 systems revealed a two-band pattern, while only one band was generated in the ME enzyme in the E. granulosus s.s. genotype. In the E. canadensis, the MDH and NH1 enzymes showed one band, and the ME enzyme represented a two-band pattern. Conclusion: Our findings suggest that E. granulosus s.s. and E. canadensis genotypes have entirely different isoenzyme patterns for NH1, G6PD, MDH, and ME.


INTRODUCTION
ystic echinococcosis is a cosmopolitan parasitic zoonosis caused by E. granulosus s.l. [1] . Globally, CE is ranked second on the list of food-borne parasitic diseases and also among the 17 most neglected tropical diseases prioritized by the WHO [2] . According to WHO report [2] , more than one million people are affected by echinococcosis at any given time [3] .
The definitive host of E. granulosus is domestic dogs, other canids, or even lion. A wide range of ungulates, including sheep, goat, cattle, and camel, plays as intermediate host. Humans acts as aberrant, dead-end intermediate hosts [3] . Contaminated food and drinking water or exposure to the contaminated environment are likely the sources of infection for humans [3] . CE is widespread and common in different regions of the world, especially the Middle East, India, South America, and Australia [4] . It is very important to study different aspects of the disease since Iran is one of the endemic regions, with ~1% of its total surgeries related to this disease [5] .
Considerable molecular studies on CE have been carried out in Iran during recent years [6,[10][11][12][13][14][15] . Based on the previous studies, E. granulosus s.s. and E. canadensis have been considered prevalent genotypes in different parts of Iran [6,12,16,17] . Moreover, a number of protein studies related to E. granulosus have been conducted in recent years in Iran [15,[18][19][20][21] . The present study aimed to investigate the isoenzyme pattern of the larval stage of E. granulosus s.s. and E. canadensis genotypes and enzyme pattern changes within each one of these genotypes and between these two genotypes in Iran. As there is no phenotypic study on the isoenzyme pattern of the E. granulosus parasite in Iran, the current research work is the first in this field.

Sample collection
CE cysts were isolated from the Central, Western, Eastern, and Southern parts of Iran. Animal specimens (sheep, cattle, and camels) were collected from Shiraz, Ilam and Birjand industrial slaughterhouses during May 2018 through December 2020. Human CE cyst were isolated from different hospitals in Shiraz and Tehran and transferred to the Parasitology Laboratory at the Medical School, Shiraz University of Medical Sciences. Following the centrifugation of all samples, a total of 30-100 µl of deposited protoscoleces were removed and washed three times with PBS. The obtained sediments were finally kept at -21 °C for subsequent experiments.

DNA extraction
DNAs were extracted from all collected samples using a DNA extraction kit (Bioneer Company, South Korea) following the manufacturer's instructions.

DNA sequencing method
All sequences were compared pairwise with each other, and the sequences were deposited in the GenBank using the program nucleotide BLAST of the NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Equal length for each sequence was trimmed with Bioedit software v.7.2 (https://bioedit.software.informer.com/ 7.2/), and global sequence alignments were performed using the ClustalW algorithm. A phylogenetic analysis was carried out on the sequence data obtained in the  present study, and the data were compared with other species/genotypes of Echinococcus. The best-fit nucleotide substitution model and phylogenetic tree were generated using Mega 6 software (https://www. megasoftware.net).

Enzyme extraction
The method of comparing isozymes was performed based on approaches used previously in 2020 [23,24] . The sediments of samples were thoroughly mixed with equal volumes of enzyme stabilizers (2 mM of dithiothreitol, aminocaproic acid, and EDTA solution). The samples were freezed and thawed at -196 °C and 25 °C, respectively for 10 times and then transferred to 0.5-ml tubes. The samples were then centrifuged at 15,000 ×g at 4 °C for 80 min, and the supernatant solution containing water-soluble proteins was kept at -70 °C until use.

Isoenzyme electrophoresis
The discontinuous polyacrylamide gel electrophoresis method was applied to perform the isoenzyme electrophoresis by vertical electrophoresis [25] . Electrophoresis was performed using the stacking gel of 4% and separating gel of 8%. In the present study, we assessed the activities of five enzymatic systems, including G6PD, ME, ICD, MDH, and NH1. The RF or relative migration distance, the number, and frequency of isoenzyme banding patterns or zymodeme of each genotype were determined and compared with each other. To determine the RF value of each band, we divided the distance traveled by the isoenzyme band from the origin by the length of the gel. Cocktail staining for visualizing each enzyme in polyacrylamide gel is shown in Table 2.

RESULTS
A total of 32 CE cyst isolates were isolated from 8 humans and 24 animals (sheep, cattle, and camels). The collected samples were characterized by PCR and then sequencing as E. granulosus s.s. (24 isolates) and E. canadensis (8 isolates) genotypes ( Table 3).

Results of PCR for cox1 primers
Agarose gel electrophoresis for cox1 primers is depicted in Figure 1.

Results of DNA sequencer technique and phylogenetic tree analysis
Samples were sent to Takapouzist Company for identifying the DNA sequence. The results showed that E. granulosus s.s. (G1) was present in 75% of the samples, and 25% were E. canadensis (G6). The phylogenetic tree was constructed using the evolutionary distances computed using the Maximum Composite Likelihood method (UPGMA tree) in the MEGA 6.0 version ( Fig. 2 and Table 4).

Isoenzyme patterns of G6PD, ME, MDH, NH1, and ICD
The isoenzyme patterns of G6PD, ME, MDH, and NH1 revealed two zymodemes. Zymodeme 1 was observed in 24 isolates, all of which were E. granulosus s.s. genotype, and zymodeme 2 was detected in 8 isolates that were E. canadensis genotype (Fig. 3). The ICD isoenzyme pattern also demonstrated two zymodemes, zymodeme 1 in 26 isolates (81.25%) and zymodeme 2 in 6 (18.75%) isolates that were E. granulosus s.s. and E. canadensis genotypes respectively (Fig. 3E). The number of bands and RF in each enzymatic system is shown in Table 5.

DISCUSSION
The present study investigated the isoenzyme pattern and molecular characterization of the E. granulosus s.l. larval stage in different parts of Iran.
The result showed that E. granulosus s.s. and E. canadensis genotypes had entirely different isoenzyme patterns in four enzyme systems (NH1, G6PD, MDH, and ME) used in this study. However, in the ICD enzyme system, it was impossible to differentiate between E. granulosus s.s. from E. canadensis strains. In the E. granulosus s.s. strain, MDH and NH1 revealed a two-band pattern, whereas the ME enzyme had a single main band. This trend for E. canadensis strain was reverse. Both genotypes studied in ICD and G6PD enzyme systems had the same number of bands with different RF values. The provided isoenzyme data represent a high degree of homogeneity within the studied strains (E. granulosus s.s. and E. canadensis strains). In this research, the isolates of each species showed similar iso-enzymatic patterns, and the studied enzymatic systems clearly showed the difference between the two species. Our results highlight the importance of using several different criteria when attempting to fully characterize an intra-strain differentiation of E. granulosus in any geographical locality and from a particular intermediate (or definitive) host. These criteria include ecological, immunological, morphological, biochemical, isoenzymatic, and in vitro developmental studies [26] . The study performed by Haag et al. [27] , revealing E. granulosus intra-strain polymorphisms, reported limited or no variation within the four studied isolates; this finding is consistent with the isoenzyme pattern reported by other researchers [25,27] .
The results obtained in the current study regarding the ability of the zymodeme technique to differentiate between E. granulosus genotypes are in linem with those reported by other studies in different geographical areas [26,28,29] . Turčeková et al. [30] proved the suitability of GPI and MDH enzymes for discriminating G7 and G1 of E. granulosus strains. Hosseini et al. [31] investigated the isozyme pattern of G6PD and ICD in E. granulosus s.s. native to Iran and detected two different strains, sheep-dog (genotype G1) and camel-dog (genotype G6) of this parasite [31] . Siles-Lucas and Cuesta-Bandera [32] showed that the SDS-PAGE technique for differentiating between the various E. granulosus strains was inappropriate. However, we found out that zymogram analysis of E. granulosus extracts using discontinuous SDS method was suitable to identify different E. granulosus strains. Iran. Biomed. J. 27 (2 & 3): 136-145 We are actually examining a parasite's phenotypic characteristics when we compare the isoenzymes of various parasite genotypes, and because these phenotypic characteristics accurately depict the genotype, the results can be taken to be in agreement with one another. In a previous electrophoretic study, Le Riche and Sewell [33] suggested that GPI isoenzyme profiles of E. granulosus belonging to UK sheep and cattle are very similar. However, these patterns are different from those produced by hydatid material obtained from two infected Nigerian camels. Isoenzyme technique as a relatively inexpensive method is still a marker with perfectly-known Mendelian inheritance, which permits multilocus analysis and is applied for most living organisms. Population genetics and phylogenetic research benefit from the use of two different types of genetic markers since they can examine various regions of the genome and have different evolutionary patterns [34] . In addition to molecular and pharmacological approaches, proteomic approaches play a significant role in identifying several excretory-secretory proteins. The ability to fully comprehend the connection between Echinococcus and its host and the mechanisms mediated by them is made possible by the identification of the proteins present in hydatid fluid. Many of these proteins may only or mostly be expressed by specific isolates and species; therefore, proteomic information is also very useful in interpreting the results of molecular and biochemical investigations [35] .
The method of determining isoenzyme characteristics in many microorganisms, such as Leishmania parasite species, has been proposed as a gold standard and as a repeatable and very accurate phenotypic method for identifying other infectious disease agents, which is of interest to researchers and scientists. Despite the emergence of new techniques, such as proteomics and genome-based methods, the mentioned phenotypic method still maintains its value. However, our study has some limitations, including the small number of enzyme systems examined and the lack of E. granulosus larval stage isolates from other intermediate hosts, such as goat, in Iran.
The findings of the present study show that the E. granulosus s.s. and the E. canadensis genotypes have entirely different isoenzyme patterns in the enzymes of NH1, G6PD, MDH, and ME. The importance of isoenzyme method increases when the difference between two genotypes is clearly shown by using a limited number of enzyme systems. Such precision in distinguishing two different organisms that are macroscopically similar has a great advantage in biology.

DECLARATIONS Acknowledgments
This article has been derived from a Ph.D. thesis (number: 20037) at Shiraz University of Medical Sciences, Shiraz, Iran. We would like to thank the cooperation of all the research centers and units.

Ethical statement
The protocol of this study was approved by the Ethics Committee of the Shiraz University of Medical Sciences, Shiraz, Iran (ethical code: IR.SUMS.REC. 1399.378).

Data availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Author contributions
MD: formal analysis and investigation, methodology, and writing original draft preparation; SMS: conceptualization and Writing-review and editing; RS: formal analysis and investigation; AV: methodology; AR: methodology; GRH: conceptualization, writingreview and editing, funding acquisition, resources, and supervision.

Conflict of interest
None declared.

Funding/support
No fund received.